Hubert A. Eddy

Resistance to adriamycin in multicellular spheroids

Abstract Spheroids of EMT-6 mammary tumor cells were markedly more resistant to different exposure doses of Adriamycin (ADR) than monolayer cells in exponential or plateau growth phases. For example, after 1 hr exposures to 0.5 μ/ml, surviving fractions determined by colony formation assay were approximately 0.3 for cells from spheroids treated intact and 0.001 for single exponential phase cells. To evaluate whether this resistance was related to poor drug uptake the distribution of the natural fluorescence of ADR equivalents was determined fluorimetrically and by direct microscopic observations. A concentration gradient of fluorescence was observed from the outside to the centers of spheroids even after high concentrations (10 μ/ml) and long exposure times (2 hrs). Cells from dissociated spheroids took up more drug than intact spheroids further indicating the existence of a significant diffusion barrier. When the surviving fraction of cells was plotted versus absorbed ADR equivalents the cells in intact spheroids were still more resistant and both curves were bicomponent with the most resistant fraction comprising about 20% of the cells. By using a selective disaggregation technique after intact spheroids had been exposed to the drug it was possible to show directly that the inner spheroid cells were most resistant (D 0 = 0.25 μ/10 6 cells). This resistance was not due to differences in the cell cycle state of these inner cells since separate experiments showed that both exponential and plateau phase monolayer cells were about equally sensitive when the surviving fraction was plotted vs absorbed drug (D 0 , = 0.04 μ/10 6 cells). Thus, other factors related to the metabolic state of the cells, the microenvironment, or the formation of different drug products must account for the observed resistance. Pretreatment of spheroids with misonidazole before ADR effectively reduced this resistant population of cells.

Development of the vascular system in the hamster malignant neurilemmoma

Abstract The development of the vasculature in the hamster malignant neurilemmoma was studied during growth in the transparent cheek pouch chamber. Prominent features in this process are: (1) Progressive dilatation, tortuosity and some bulbous formations in host venules in the immediate vicinity of the tumor implant. Occasional focal dilatation of some host arterioles. (2) Small focal erythrocytic extravasations. (3) Sprouting of host venous vessels in the perimeter of the implant and subsequent anastomosing of these sprouts and/or their branches to form loops directed toward the center of the implant. Continued sprouting, in effect, produces remodeling of the host venules. (4) Establishment of the tumor capillary net in 4–7 days. The bed assumes a dense net-like arrangement of short, profusely anastomosing, thin-walled endothelial tubes which conduct blood at a brisk rate. (5) Progressive increase in capillary caliber during tumor growth. (6) Prominent focal or general reduction in blood flow after about 10 days of growth. This appears related to the development of tissue growth pressure within the rigid cheek pouch chamber.

Effect of hyperthermia, radiation and adriamycin combinations on tumor vascular function☆

Pathophysiologic studies of tumor vascular responses to hyperthermia, radiation or adriamycin given alone or in specific combinations have been made in the cervical carcinoma grown in the transparent cheek pouch chamber of the Syrian hamster. A specially designed chamber containing a compartment for flowing water enabled controlled heating of the tumor and pouch to within 0.2 degrees C; the desired temperatures were achieved within one minute. Heating at 42 degrees C for 30 minutes was followed, at 1, 5 or 24 hours, by a second heating for 30 minutes at 42 degrees C. In addition, the same period of heating was preceded or followed, at 1, 5 or 24 hour intervals, by a single exposure to 2000R or a single intravenous injection of adriamycin given at a rate of 0.45mg/100gm body weight. Of the three modalities, heat appeared to have the greatest acute effect on the tumor vascular system. A single dose of heat produced a rapid but transient constriction followed by a prominent dilation of vessels. Two heating periods given at a 1 hour interval caused persistent stasis in the tumor which progressed to coagulation necrosis. Although heating prior to irradiation or adriamycin, in general, increased the vascular responses to these agents, this sequence gave no tumor control. Radiation or adriamycin given prior to heating had relatively little effect on the vascular response to heating and produced no tumor control except when heat was applied shortly after irradiation. These studies indicate that changes in the microvasculature and perfusion in tumors, in response to hyperthermia alone or combined in specific sequences with radiation, can alter the internal environment of the tumor to produce a greater degree of tumor control than can be attributed to direct cell killing by these agents.

Microangiographic techniques in the study of normal and tumor tissue vascular systems

Abstract Techniques are described for the visualization of either the arterial or the entire vascular system of normal or malignant tissues. Such techniques are useful for evaluating changes in large areas of a vascular bed following a variety of experimental treatments. Red lead oxide or Pelikan ink suspended in a heparinized gelatin-Joy detergent medium can be visualized in perfused vessels by high resolution microradiography or photomicrography. The system facilitates the evaluation of pathologic lesions with regard to the vascular integrity of the organ or tissue under investigation.

Tumor vascular responses following irradiation

Abstract Vascular responses of the hamster malignant neurilemmoma, grown in the transparent cheek pouch chamber, were studied following single 3000-R exposures to ionizing radiation. Variable degrees of edema and extensive reduction in blood flow occurred during the first 24–30 hr, with subsequent restoration toward normalcy accompanied by small focal hemorrhaging. Inhibition of both tumor growth and neovascularization occurred during the first 3 to 5 days. Subsequent regrowth, as indicated by newly vascularizing foci of tumor cells, occurred earliest in the perimeter of the tumor where radiosensitivity is thought to be greatest. Return of cellular proliferation within the tumor was characterized by progressive increase in caliber of persisting tumor capillaries and the formation of a new capillary network. There was no obvious correlation between the changes observed and the probability of tumor regression or regrowth.

Biological Properties and Radiation Responses of the Hamster Uterine Cervical Squamous Cell Carcinoma

Results of initial studies of the growth characteristics, immunogenicity, histopathology, and radiation response of a uterine cervical squamous cell carcinoma of the Syrian hamster are presented. The cervical carcinoma is a relatively loosely knit, undifferentiated, highly cellular tumor without a recognizable architectural pattern but with a gradation in cellular density decreasing from the perimeter to the center of the tumor. The vascular network corresponds with the histologic features such that radially arranged capillaries join a dilated, tortuous network at the perimeter and with a sparse network in the center of the tumor. Tests of the immunogenicity of the cervical carcinoma by either suppressing the immune mechanism (s) or sensitizing the animal to the tumor, indicate that this tumor provides little or no stimulus to the host immunologic system. After radiation exposures ranging from 1000 to 7000 R, tumor volume in persisting tumors shows a pattern of retardation and/or regression followed by regrowth which correlates with exposure. Increasing tumor control occurs with exposures of 3000 R and higher producing a TCD50/120 days of 5800 =L 1100 R. The cause of death in animals containing persisting tumors could be attributed to extensive pulmonary metastases and/or intercurrent infection relative to ulceration and necrosis of the tumor. The major advantages of the use of the Syrian hamster and the cervical carcinoma as a model system and as an interface for the comparison of data from other established tumor models with that from transplantable xenogenic tumor systems is also presented.

Urine output, saline intake, and adrenal factors after lethal whole-body or intestinal irradiation.

Daily urine output and saline intake were measured up to 5 days after whole-body or intestinal x-irradiation (1460 R) of intact or adrenalectomized rats treated or not with adrenal cortical extract...

The response of Syrian hamster sarcomas to single exposures of X rays.

Abstract Three types of Syrian hamster tumors (reticulum-cell lymphosarcoma, fibrosarcoma, and malignant neurilemmoma) were studied after transplantation into the cheek pouch. There was a transient delay in growth or decrease in volume of all tumors at all levels of exposure. Recovery of primary tumor growth was inversely related to exposure level. Frequency of tumor control increased and incidence of metastases decreased with increasing exposure level. Based upon these parameters, fibrosarcoma is most sensitive to single brief x-ray exposures, followed by malignant neurilemmoma and reticulum-cell lymphosarcoma.